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Basic Research Paper

Inhibitory effect of wild-type p53 gene on excessive replication of centrosomes in leukemia cell line K562

Wen-Jun Tian, Wen-Li Feng, Hong-Bin Wang, Shi-Feng Huang, Wei-Xi Cao and Zong-Gan Huang

Background and Objective: Mutation and deletion of p53 gene in tumor cells is one of the major reasons for aneuploid development and genomic instability. Abnormal centrosomes exist in chronic myelogenous leukemia patients at different clinical stages; the degree of abnormality is associated with clinical stage and more severe in blast crisis stage. This study was to establish the leukemia K562 cell line with exogenous wild-type p53 gene, and explore the effect of p53 gene on centrosomes in K562 cells. Methods: The recombinant adenoviruses carrying wild-type p53 gene (Ad5wtp53), mutant P53 gene (Ad5mtp53) and green fluorescent protein gene (Ad5GFP) were amplified respectively in HEK293 cells, and co-infected with cation polybrene into K562 cells respectively; uninfected K562 cells were used as blank control. The infection efficiency was analyzed by flow cytometry. P53 expression was detected by Western blot. Centrosomes were counted under the laser confocal microscopy after indirect immunofluorescence staining. The expression of Gadd45a (growth arrest and DNA damage), BubR1 (Bub 1 related), and Aurora A was detected by Western blot. Results: K562 cell line with exogenous wild-type p53 gene was established. The infection efficiencies of three groups were over 60%, and P53 sustained to express for 72 h. The percentage of cells with amplified centrosomes (more than 2/cell) in Ad5wtp53 group was decreased to (0.38 ± 0.02)%, lower than that of blank control group (p < 0.05). Meanwhile, the protein levels of Gadd45a and BubR1 in Ad5wtp53 group were upregulated by 93% and 88% of blank control (p < 0.05), and the protein level of Aurora A was deregulated by 56% of blank control (p < 0.05). Conclusions: P53 protein is sustained to express in K562 cells after being infected by Ad5wtp53. Wild-type p53 can suppress excessive replication of centrosomes that might contribute to the up-regulation of Gadd45a and BubR1 protein expression as well as the downregulation of Aurora A protein expression.

Authors

Wen-Jun Tian

Chongqing University of Medical Sciences; Chongqing, China

Wen-Li Feng Corresponding author: fengwlcqmu@sina.com

Chongqing University of Medical Sciences; Chongqing, China

Hong-Bin Wang

Chongqing University of Medical Sciences; Chongqing, China

Shi-Feng Huang

Chongqing University of Medical Sciences; Chongqing, China

Wei-Xi Cao

Chongqing University of Medical Sciences; Chongqing, China

Zong-Gan Huang

Chongqing University of Medical Sciences; Chongqing, China